Role of Glycans in Human Embryonic Stem Cell Conversion to Neural Precursor Cells

Role of Glycans in Human Embryonic Stem Cell Conversion to Neural Precursor Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00200
Award Value: 
$708,000
Stem Cell Use: 
Embryonic Stem Cell
Status: 
Closed
Public Abstract: 
Like a thick frosting on a cake, complex sugar chains decorate every surface of every cell. Try to approach a cell, as friend or foe, and the canopy of sugars is the first gate-keeper. Each cell makes and organizes these sugar chains, called glycans, on its surface. They are very complicated molecules, and different cells choose to decorate themselves with different glycans—for reasons best known to the cells themselves. Because glycans have such complicated structures, it is hard to work with them and understand their function. They are much more diverse than DNA and proteins, and so the technology for dissecting their structures and functions has lagged behind the others in the molecular revolution in biology and medicine. Glycans are complicated molecules, hard to work with, difficult to understand, but they are absolutely indispensable to life. Human genetic disorders where just one step in their assembly is missing causes mental retardation, seizures, blindness and poor motor skills. Glycans are used for communication both within and between cells, and this is especially true when cells signal each other about their past and future journeys within the developing body and exactly where they will go and what they will become during development. Embryonic stem cells have a particular set of glycans on their surface and change as the cells develop into different cell types. What directs these changes? Are they all important? Can we manipulate a cell’s fate or convince it to behave in a certain way by changing—or maintaining—the sugar coating? The scientific literature shows that changing surface sugar chains can have profound effects.New technology in the field of “Glycobiology” makes it possible to analyze minute amounts of material with great precision and define these structures. Thanks to our collaborators, we can produce substantial amounts of human embryonic stem cells that uniformly transition into neural precursor cells. Our plan is to describe in detail these glycan changes as they occur and then determine which are actually essential for cells to reach that point. How do these glycans allow them to go further on to neurons, oligodendrocytes and astrocytes? We hope to exploit these unique sugar signatures to identify and isolate cells that will have a particular developmental fate. This is only the beginning. It is a catalog of events and a parts list, but we know how the parts are assembled and what machines are needed. Since we are only beginning the stem cell enterprise, it’s important to define these elements from the beginning. We hope to use this knowledge to direct and influence stem cells to travel down the paths we prefer, since we already know the path is sugar coated and the coating is essential.
Statement of Benefit to California: 
The necessary existence of the CIRM is the largest benefit for the state and demonstrates our commitment to the concept that research and understanding are the keys to a better life for all citizens. By understanding how the NPC’s differentiate and which glycans are important, we may be able to forecast which genes will be likely to cause developmental problems if they contain specific polymorphisms. As individual genome scans for disease susceptibility become more commonplace in the community, we will need to identify those genes. Localized manipulation of the cell surface glycans on injured nerve cells may help stimulate their growth. This has already been seen using injected chondroitinase and sialidase digestions to stimulate robust nerve growth in mechanically injured, debilitated rodents. This specific project benefits the state economically. Success in recognizing specific cell types during the differentiation enables the development of reagents and assay kits to isolate such cells, scaling up the isolation, and adapting the lectin-binding purification concept to other types of differentiating cells. The demand for more therapeutic use of stem cells will require an industry prepared to identify and fractionate those cells with special surface properties, some of those being defined by lectin binding.
Progress Report: 

Year 1

Complex sugar chains decorate the cell surface. The cell makes and organizes these complicated molecules on their surface, for reasons best known to the cells themselves. We want to understand their function of sugar chains (glycans). We know they help cells communicate about their past and future journeys within the developing body, exactly where they go and what they become during development. Human genetic disorders where just one step in their assembly is missing, and mental retardation, seizures, blindness and poor motor skills often result. Embryonic stem cells have a particular set of glycans on their surface and it changes as the cells develop into different cell types. We have two major goals in this project. First, to identify the human embryonic stem (hES) cell glyco-signatures and then determine what changes as they differentiate into neural precursor cells (NPC). Second, to ask whether these glycan changes are important for correct timing and normal differentiation of the cells. What happens if we change or keep the original sugar coating? Does it change the cell’s fate? We can produce substantial amounts of hES cells that uniformly transition into NPC. Because these are early days of understanding stem cells, it was important to analyze different hES cell lines that differentiate into NPC under two different conditions. Glycan analysis can be done indirectly using sugar-binding proteins called lectins, or directly by mass spectrometry of glycans released from proteins or lipids. Another way to infer an alteration in glycan composition is by changes in the expression of genes encoding enzymes that make glycan chains. This is called transcriptional profiling. If more glycan is needed, more enzyme may be needed to produce that glycan; less glycan involves reduced expression of those genes. Sometimes more than one enzyme (gene product) carries out the very synthetic same step, but one of the enzymes may prefer a certain kind of glycan over another. We used both approaches (lectin binding and transcriptional profiling) in parallel. Lectins bind to various types of N- and O-linked glycans and we used an instrument that detects small changes in the binding of cell fragments to a battery of 40 lectins. Carefully worked out and standardized conditions showed that only a few lectins consistently increased binding during transitions from hES cells into NPC. These lectins recognized some changes, but were in the 2-3 fold range at most. Comparison of hES cells grown on mouse feeder-layers to those grown without feeders, showed mostly similar (not identical) patterns. The changes were subtle, so instead of continuing glycan analysis by mass spectrometry, we chose to analyze transcriptional profiles. Our goal was to identify genes whose expression changed quite substantially. Initially, we used results obtained by Dr. Terskikh to profile the entire genome when hES cells developed into NPC. Here, subtle changes in glycosylation gene expression (2-3 fold increases/decreases) were near baseline making it hard to know if they were accurate. An exception was the chondroitin sulfate core protein, decorin, which increased >30-fold during differentiation. Since many glycosylation-related genes are expressed at low levels, we went to colleagues at the University of Georgia for analysis of >700 genes using Real Time PCR. Their 5 order of magnitude dynamic range and reproducibility of triplicate samples (+/-15-20%) is impressive. These results showed no (<2-fold) changes in sugar precursor metabolism, N-linked glycan precursor synthesis, and only a decrease in tetra-branched chains N-glycans. Considerable increases (5->100 fold) in terminal modifications common to both N- and O-linked glycans (Sda, polysialic acids, Lewisa) in the different hES cells prepared by either method. Fringe genes encoding glycans that modify Notch signaling (LFNG, especially) increased. GPI anchor biosynthesis gene that adds a final mannose (PIGZ) is decreased as is palmitoylation of the anchors. Fine differences in HS-6 sulfation changed and one especially prominent gene that appears only in neural tissue (NDST4) showed a substantial increase. These transcriptional changes are more wide-ranging and different than what we could see using lectin profiles or direct analysis of N- and O-glycans. The two approaches do not necessarily give congruent results. These transcriptional changes allow us to focus on genes more likely to be affected by the knockdown strategies we planned for aim two. In that aim, we want to prevent the up-regulation of those specific genes to see if it affects differentiation. If preventing transcriptional increase (by siRNA) does have developmental consequences then we will be in a better position to analyze which specific glycans are important. Our top knockdown candidates are: Decorin, Sd antigen (B4GALNT2), polysialic acid (STaSia1, 2, 3), NDST4 and Lewis antigen (FT3). We hope to cover these in the no-cost extension.

Year 2

Complex sugar chains decorate the cell surface. The cell makes and organizes these complicated molecules on their surface, for reasons best known to the cells themselves. We want to understand their function of sugar chains (glycans). We know they help cells communicate about their past and future journeys within the developing body, exactly where they go and what they become during development. Human genetic disorders where just one step in their assembly is missing, and mental retardation, seizures, blindness and poor motor skills often result. Embryonic stem cells have a particular set of glycans on their surface and it changes as the cells develop into different cell types. We have two major goals in this project. First, to identify the human embryonic stem (hES) cell glyco-signatures and then determine what changes as they differentiate into neural precursor cells (NPC). Second, to ask whether these glycan changes are important for correct timing and normal differentiation of the cells. What happens if we change or keep the original sugar coating? Does it change the cell’s fate? We can produce substantial amounts of hES cells that uniformly transition into NPC. Because these are early days of understanding stem cells, it was important to analyze different hES cell lines that differentiate into NPC under two different conditions. Glycan analysis can be done indirectly using sugar-binding proteins called lectins, or directly by mass spectrometry of glycans released from proteins or lipids. Another way to infer an alteration in glycan composition is by changes in the expression of genes encoding enzymes that make glycan chains. This is called transcriptional profiling. If more glycan is needed, more enzyme may be needed to produce that glycan; less glycan involves reduced expression of those genes. Sometimes more than one enzyme (gene product) carries out the very synthetic same step, but one of the enzymes may prefer a certain kind of glycan over another. We used both approaches (lectin binding and transcriptional profiling) in parallel. Lectins bind to various types of N- and O-linked glycans and we used an instrument that detects small changes in the binding of cell fragments to a battery of 40 lectins. Carefully worked out and standardized conditions showed that only a few lectins consistently increased binding during transitions from hES cells into NPC. These lectins recognized some changes, but were in the 2-3 fold range at most. Comparison of hES cells grown on mouse feeder-layers to those grown without feeders, showed mostly similar (not identical) patterns. The changes were subtle, so instead of continuing glycan analysis by mass spectrometry, we chose to analyze transcriptional profiles. Our goal was to identify genes whose expression changed quite substantially. Initially, we used results obtained by Dr. Terskikh to profile the entire genome when hES cells developed into NPC. Here, subtle changes in glycosylation gene expression (2-3 fold increases/decreases) were near baseline making it hard to know if they were accurate. An exception was the chondroitin sulfate core protein, decorin, which increased >30-fold during differentiation. Since many glycosylation-related genes are expressed at low levels, we went to colleagues at the University of Georgia for analysis of >700 genes using Real Time PCR. Their 5 order of magnitude dynamic range and reproducibility of triplicate samples (+/-15-20%) is impressive. These results showed no (<2-fold) changes in sugar precursor metabolism, N-linked glycan precursor synthesis, and only a decrease in tetra-branched chains N-glycans. Considerable increases (5→100 fold) in terminal modifications common to both N- and O-linked glycans (Sda, polysialic acids, Lewisa) in the different hES cells prepared by either method. Fringe genes encoding glycans that modify Notch signaling (LFNG, especially) increased. GPI anchor biosynthesis gene that adds a final mannose (PIGZ) is decreased as is palmitoylation of the anchors. Fine differences in HS-6 sulfation changed and one especially prominent gene that appears only in neural tissue (NDST4) showed a substantial increase. These transcriptional changes are more wide-ranging and different than what we could see using lectin profiles or direct analysis of N- and O-glycans. The two approaches do not necessarily give congruent results. These transcriptional changes allow us to focus on genes more likely to be affected by the knockdown strategies we planned for aim two. In that aim, we want to prevent the up-regulation of those specific genes to see if it affects differentiation. If preventing transcriptional increase (by siRNA) does have developmental consequences then we will be in a better position to analyze which specific glycans are important. Our top knockdown candidates are: Decorin, Sd antigen (B4GALNT2), polysialic acid (STaSia1, 2, 3), NDST4 and Lewis antigen (FT3).

© 2013 California Institute for Regenerative Medicine